Aptamers, derived from the latin aptus, meaning, ‘to fit’, are oligonucleotides that have a specific three dimensional shape and consequent biological activity. Aptamers are generally produced through a process named “systematic evolution of ligands by exponential enrichment” or “SELEX,” which is an iterative selection and amplification process. Nucleic acids that bind to a target are selected (non-binders are simply washed away) and then subjected to a round of amplification. As this process is iterated, tightly binding aptamers are enriched in the population, and extremely tight and specific binding between the aptamer and the target can be achieved. The reader is referred to U.S. Pat. No. 5,270,163 and the very large family of related patents for detailed SELEX protocols.
The extraordinary capacity of aptamers to bind tightly to specific targets underlines their tremendous potential as molecular therapeutics. For example, aptamers can be used to selectively target cells (such as tumor cells or pathogens) for death.
For example, U.S. Pat. No. 6,566,343 discusses the potential for aptamers directed at cell surface components of bacteria, cancer cells and parasites to activate the complement system and bring about the lysis of target cells. The patent discloses the linkage of two aptamers—one directed against the target cell and a second one against a component of the complement system (thus recruiting the complement cascade to the target cell)—to achieve complement activation and targeted cell death.
There are two distinct disadvantages to this approach. First, the aptamer-aptamer conjugates are subject to degradation from serum nucleases and second, the aptamer-aptamer conjugates are subject to rapid clearance by the kidneys. Thus, although aptamers are a powerful targeting system, in vivo nucleic acid stability remains a problem.
A Canadian team of researchers (Dougan et al., 2000) demonstrated that 3′-biotinylation of DNA significantly increased its resistance to serum nuclease activity. This was presumably due to steric hindrance and suggests that any 3′ or 5′ capping or nucleic acid modification should improve nucleic acid stability in vivo.
However, our research surprisingly indicates that 5′-biotinylation is not very effective against serum degradation of DNA, nor is the incorporation of 2′-Fluoro modified deoxynucleotide triphosphates (2′F-dNTPs). Thus, the stability issue is not as simply addressed as one might predict. Hence, improved methods of stabilizing nucleic acids for in vivo therapeutic use are still needed and the invention addresses this problem.